(1) In the absence of calmodulin, the mechanism of calcineurin activation by Ni(II) is different from that in the presence of calmodulin. Two Ni(II) ions can bind to the enzyme, one leads to activation, the other to deactivation. However, both the free enzyme and the activated enzyme-Ni(II) complex appear to be thermally less stable than the enzyme-two Ni(II) complex. (2) At least two forms of Ca(II)-inhibited phosphatases from bovine brain have been identified. A 52,000 molecular weight form was found to catalyze only non-protein phosphates. Another form was found to utilize histone H2B as substrate. (3) The intrinsic Ca(II) dissociation constants for the four binding sites of calmodulin have been determined according to the latest model based on x-ray crystallography, NMR, and Ca(II) binding data. The values of the constants are consistent with the existence of two Ca(II)-binding domains: A high-affinity domain with two highly cooperative binding sites and a low-affinity domain with two less cooperative binding sites. When combined with the target enzyme, the low-affinity sites must undergo dramatic conformational change and acquire much higher affinities for Ca(II). (4) Model studies using the Fe(II)- ferrozine system to examine the theory and practice of determination of binding stoichiometry by the continuous variation method (the Job plot) revealed that (a) the system can be best described by a one-step or infinite cooperativity mechanism in which three molecules of ferrozine combine with one Fe(II), and that (b) at low total molar concentration of the two reactants, when the Fe(II) concentration is expressed in terms of binding sites, anomalous stoichiometries may be obtained. The apparent stoichiometry increases with decreasing total molar concentration and varies between 1 (the correct value) and 3.